siRNA against presenilin 1 (PS1) down regulates amyloid β42 production in IMR-32 cells

Background  

One of the pathological hallmarks of Alzheimer's disease (AD) is the deposition of the ~4 kDa amyloid β protein (Aβ) within lesions known as senile plaques. Aβ is also deposited in the walls of cerebral blood vessels in many cases of AD. A substantial proportion of the Aβ that accumulates in the AD brain is deposited as Amyloid, which is highly insoluble, proteinaceous material with a β-pleated-sheet conformation and deposited extracellularly in the form of 5-10 nm wide straight fibrils. As γ-secretase catalyzes the final cleavage that releases the Aβ42 or 40 from amyloid β -protein precursor (APP), therefore, it is a potential therapeutic target for the treatment of AD. γ-Secretase cleavage is performed by a high molecular weight protein complex containing presenilins (PSs), nicastrin, Aph-1 and Pen-2. Previous studies have demonstrated that the presenilins (PS1 and PS2) are critical components of a large enzyme complex that performs γ-secretase cleavage.     

Multi-Low-Dose Mucosal Simian Immunodeficiency Virus SIVmac239 Challenge of Cynomolgus Macaques Immunized with “Hyperattenuated” SIV Constructs

Hyperattenuated simian immunodeficiency virus SIVmac239-derived constructs Δ5-CMV and Δ6-CCI are an effort to render SIV incapable of, in practical terms, both reversion and recombination while maintaining the immune features of SIV as a retrovirus. Primary inoculation of cynomolgus macaques with 10⁸ 50% tissue culture infective doses (TCID₅₀) of Δ5-CMV or Δ6-CCI induced low-level humoral and cellular responses detectable in the absence of measureable in vivo replication. The first of three DNA boosts resulted in elevated gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) responses to Gag, Pol, and Env in the Δ5-CMV vaccine group compared to the Δ6-CCI vaccine group (P = 0.001). Weekly intrarectal challenge with a low dose of SIVmac239 followed by a dose escalation was conducted until all animals became infected. The mean peak viral load of the Δ5-CMV-vaccinated animals (3.7 × 10⁵ copies/ml) was ∼1 log unit lower than that of the control animals. More dramatically, the viral load set point of these animals was decreased by 3 log units compared to that of the controls (<50 versus 1.64 × 10⁴ copies/ml; P < 0.0001). Seventy-five percent (6/8) of vaccine recipients controlled virus below 1,000 copies/ml for at least 6 months, with a subset controlling virus and maintaining substantial CD4 T-cell counts for close to 2 years of follow-up. The correlates of protection from SIV disease progression may lie in the rapidity and protective value of immune responses that occur early in primary SIV infection. Prior immunization with hyperattenuated SIVmac239, even if sterilizing immunity is not achieved, may allow a more advantageous host response.To date, the most promising approach to inducing sterilizing immunity in the macaque model has been through the use of live attenuated virus (LAV) vaccines based on simian immunodeficiency virus (SIV). A major advantage of an attenuated virus strategy for the development of a human immunodeficiency virus (HIV) vaccine is the ability of attenuated viruses to induce broad and persistent immunity (29, 51). In particular, SIV strains engineered with deletions of nef (SIVΔnef) have afforded the most significant protection upon challenge with pathogenic SIV (13, 14, 29, 60, 65, 72). Numerous SIV-derived live attenuated vaccine models have been developed, many of which employ deletions in the viral accessory genes (3, 12, 14, 15, 25, 29, 30, 53, 64, 72). In many cases, vaccinations have been shown to substantially decrease viral burden during the acute phase of infection, maintain low to undetectable levels of virus during the chronic phase of infection, and limit the progression to AIDS. Although promising, a major caveat to the live attenuated virus vaccine approach is the potential for compensatory reversion and the observations that incompletely attenuated viruses may harbor residual pathogenicity (5, 10, 14). Even SIV constructs containing multiple deletions in nef, vpr, and the negative regulatory element (NRE) can cause AIDS-like disease in adult macaques and particularly in neonates (4, 5, 27, 53). This may be analogous to some human long-term nonprogressors infected by nef-deleted HIV variants in whom a slowly increasing viral burden has been accompanied by disease progression (22, 34, 37). Additional mutations can be engineered into vaccine vectors to generate highly attenuated viruses, but this often comes at the expense of their protective efficacy (8, 23, 30).We previously made two series of novel live attenuated SIV vaccine models (25) in which the simplified SIV constructs retain all the structural viral proteins but have inactivating mutations for all viral accessory genes. These constructs retain significant antigenicity, without the pathogenic effects associated with accessory viral factors, thus limiting or eliminating the potential for reversion (25).Whether administered parenterally or mucosally, conventional challenge trials in macaques have often utilized artificially high single-dose inocula in an effort to ensure that most, if not all, of the naive or placebo-immunized animal subjects become infected following a single exposure. The rationale for using a single massive challenge has been reconsidered in light of the possibility that vaccines with protective efficacy under physiologic challenge conditions may not identified. This practice is now being replaced by an approach designed to better approximate the relatively low in vivo acquisition rates following a single sexual exposure to HIV (21, 45, 69) and should provide a more realistic assessment of vaccine efficacy in “real-world” situations. Importantly, recent studies using this approach have demonstrated viremia of magnitude and kinetics comparable to that seen following single high-dose mucosal inocula (47), and this approach has been used successfully in more recent challenge trials (31, 70). Here we are assessing the safety, immunogenicity, and protective efficacy of two hyperattenuated SIV vaccine candidates following a multi-low-dose intrarectal challenge with highly pathogenic SIVmac239 in the cynomolgus macaque model.SIV-specific humoral immune responses were assessed at various time points postvaccination and postchallenge by Western blotting. Cellular immunogenicity was monitored by evaluation of peripheral T-cell responses (via gamma interferon [IFN-γ] enzyme-linked immunospot [ELISPOT] assay) following stimulation with peptide pools spanning the entire SIVmac239 proteome. The protective efficacy of the different vaccine candidates was assessed by classical endpoints, such as quantitative analysis of plasma viral load, quantitative immunophenotyping of lymphocytes, and clinical markers of disease progression. Even using extremely attenuated SIV constructs with only minimal evidence of replication, a modest immune response that can impact long-term disease progression is generated.     

Application of a new method in the study of pelvic floor muscle passive properties in continent women

The aim of this study was to present a new methodology for evaluating the pelvic floor muscle (PFM) passive properties. The properties were assessed in 13 continent women using an intra-vaginal dynamometric speculum and EMG (to ensure the subjects were relaxed) in four different conditions: (1) forces recorded at minimal aperture (initial passive resistance); (2) passive resistance at maximal aperture; (3) forces and passive elastic stiffness (PES) evaluated during five lengthening and shortening cycles; and (4) percentage loss of resistance after 1 min of sustained stretch. The PFMs and surrounding tissues were stretched, at constant speed, by increasing the vaginal antero-posterior diameter; different apertures were considered. Hysteresis was also calculated. The procedure was deemed acceptable by all participants. The median passive forces recorded ranged from 0.54 N (interquartile range 1.52) for minimal aperture to 8.45 N (interquartile range 7.10) for maximal aperture while the corresponding median PES values were 0.17 N/mm (interquartile range 0.28) and 0.67 N/mm (interquartile range 0.60). Median hysteresis was 17.24 N1mm (interquartile range 35.60) and the median percentage of force losses was 11.17% (interquartile range 13.33). This original approach to evaluating the PFM passive properties is very promising for providing better insight into the patho-physiology of stress urinary incontinence and pinpointing conservative treatment mechanisms.